Granulocyte colony stimulating factor (G-CSF) is produced by mononuclear cells and fibroblast cells. It can stimulate granulocyte to form colonies, and has a stimulating effect on neutrophils. By combining with membrane receptors of the target cells, G-CSF mainly stimulates hematopoiesis of granulocyte and also promotes multipotent hematopoietic stem cells to enter the cell cycle; promotes proliferation, differentiation and maturation of myeloid hematopoietic progenitors; and drives the release of neutrophils into the blood. Meanwhile, it increases the number of peripheral neutrophils and improves their functions, such as phagocytosis, antibody-dependent cellular cytotoxic activity against tumor cells, etc. [Metcalf, Blood 67:257 (1986); Yan et. al., Blood 84(3): 795-799 (1994); Bensinger, et. al., Blood 81(11): 3158-3163 (1993); Neben, et. al., Blood 81(7): 1960-1967 (1993)]. Therefore, recombinant granulocyte colony stimulating factor is commonly used in cancer patients subjected to radiotherapy or chemotherapy, and leukemia patients after bone marrow transplantations as an adjuvant treatment.
Human G-CSFs commonly used on the market are Neupogen and Neutrogin, and a human G-CSF derivative, i.e. Neu-wp. G-CSF derivatives or their variant proteins have also been reported in a large number of references (such as U.S. Pat. No. 5,581,476, U.S. Pat. No. 5,214,132, U.S. Pat. No. 5,362,853, U.S. Pat. No. 4,904,584). These variant proteins have multiple amino acid substitutions, which have been designed to explore more stable, more active and more suitable forms of G-CSF for clinical use.
The commercially available recombinant human G-CSF needs to be injected frequently. It can be difficult to achieve a good clinical effect because of its poor bioavailability, short half-life in the human body, and vulnerability to proteases in vivo. Research has shown that the possibility of the proteins to become drugs is greatly increased when they are conjugated with polyethylene glycol (PEG). These “PEGylated” proteins have been fully applied in clinical practices (such as Katre, Advanced Drug Delivery Systems, 10:91 (1993); Inada; et. al.; J. Bioact and Compatible Polymers; 5:343 (1990)). Polyethylene glycol-protein conjugates have not only better physical and chemical stabilities, but also better resistance to protease enzymes in vivo. In addition, as the molecular weight of the conjugates increases, the half-life of the conjugates in vivo is extended. The toxicity will be reduced because of the lower possibility to produce antibodies in vivo and the reduced volume of distribution of the conjugates as comparing with original proteins.
PEG-modified G-CSF or G-CSF variant proteins have been disclosed in numerous references, such as EP0335423, EP0401384, U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,985,265, WO0044785, WO2001051510, U.S. Pat. No. 5,824,784, etc. Specifically, among the PEG-G-CSF conjugates disclosed by U.S. Pat. No. 5,985,265, the conjugates modified at the N-terminus of G-CSF have the best biological activities both in vitro and in vivo. However, the selectivity of amino groups is poor when the modification with PEG is carried out through an acylation reaction, thus producing a mixture of G-CSF modified at various positions (amino group) by polyethylene glycol. Consequently, separations and purifications are required to obtain each monomer. The yield may be low and it can be difficult to manufacture on an industrial scale.
In U.S. Pat. No. 5,824,784, polyethylene glycol aldehydes with large molecular weights are used to modify G-CSF directly. With strict control of the pH of the reaction, relatively specific linking of the PEG to the N-terminus of protein can be achieved. However, the reaction with high selectivity uses the difference in the pKa of the side chain amino group and the pKa of the N-terminal amino group. This reaction is thus relatively difficult to control in manufacturing scales. In addition, because of the variable numbers of aldehyde groups in the polyethylene glycol aldehydes with large molecular weights in each batch, it is difficult to control the ratio between aldehydes and the proteins. Consequently, the reaction yields and production costs are difficult to control. Moreover, the different biological activities of the conjugates produced by coupling of aldehydes with different amino groups will affect the homogeneity and activity of the final product.